3D Structure Model of the Principal Neutralizing Epitope of Minnesota HIV-1 Isolate

Abstract

A hierarchical procedure, using a “bottom-up” strategy and combining (i) a probabilistic approach for estimating all possible starting structures, (ii) restrained molecular mechanics algorithms for preliminary selection of all energetically preferred conformers, as well as (iii) quantum chemical computations for refining their geometry, was used to study the structural properties of the HIV-MN neutralizing epitope in terms of NMR spectroscopy data. As a result, only one of initial structures matching the experimental and theoretical data was found to be well-ground for implementing the function of immunoreactive conformation of the virus immunogenic crown. The geometric parameters of this structure in water solution were shown to correspond to a double β-turn conformation similar to that revealed in crystal for synthetic molecules imitating the central region of the HIV-MN V3 loop. The following conclusion was drawn from the comparative analysis of simulated structure with the one computed previously: the HIV-MN immunogenic tip has some inherent conformational flexibility that manifests at the alterations of hexapeptide environment and leads to the structural transitions changing the local conformation of the stretch of interest but retaining its spatial main chain fold. As a matter of record, the high resolution 3D structure model for the HIV-MN principal neutralization site was constructed, and its geometric parameters were compared with the corresponding characteristics of conformers derived earlier for describing the conformational features of immunogenic tip of gp120 from Thailand HIV-1 isolate.

The results are discussed in the light of literature data on HIV-1 neutralizing epitope structure.     

Modeling of the 3D structure of the HIV-Haiti immunodominant epitope

The 3D structure for the HIV-Haiti immunodominant epitope was computed in terms of NMR spectroscopy data using the theoretical procedure including a probabilistic approach in conjunction with molecular mechanics algorithms and quantum chemical methods. The immunogenic crown of the virus protein gp120 was shown to form in solution a prevalent conformation whose geometric parameters match the double beta-turn IV-IV. Two structures observed in crystal were found in the ensemble of the best-energy conformations of the HIV-Haiti principal neutralizing epitope. From a comparison of simulated structures with those computed previously for the HIV-Thailand and HIV-MN isolates, it was concluded that the immunogenic tip of gp120 gives rise to similar spatial backbone forms in different HIV-1 strains but has some inherent conformational flexibility of its individual amino acid residues. The differences in local fragment structures revealed in three isolates of HIV-1 are supposed to be important for the specificity of its binding with neutralizing antibodies.     

Structure and polymorphism of the principal neutralization site of Thailand HIV-1 isolate

Refining the geometric parameters for the ensemble of conformers, derived earlier in terms of NMR-spectroscopy data for the immunogenic tip of Thailand HIV-1 isolate, was carried out by quantum chemical methods. As a result, (i) the energy characteristics of initial structures were significantly improved, (ii) their relative locations on the scale of formation heats were determined, and (iii) the energy barriers between conformers under study were computed. On the basis of all data obtained, the high resolution 3D structure model, describing the set of stable conformers and containing the biologically active conformation, was proposed for neutralizing epitope of Thailand HIV-1 isolate. The following major conclusions were made based on the analysis of simulated conformations: i) the Gly-Pro-Gly-Gln-Val-Phe stretch forming the immunogenic crown of Thailand HIV-1 isolate exhibits the properties characteristic for metastable oligopeptide that constitutes in solution the dominant structure with other conformations admissible; (ii) three structures out of five NMR-based starting models form the cluster of conformers which adequately describes general conformational features of this functionally important site of gp120; (iii) two structures residing in this cluster are found to be well-ground for implementing the function of immunoreactive conformation of the stretch of interest; (iv) in spite of this observation, the "global" structure which gives rise to inverse gamma-turn in the central Gly-Pro-Gly crest of Thailand HIV-1 gp120 is proposed to be the most probable conformation responsible for the formation of viral antigen-antibody complex in particular case under study.     

Dual spatial folds and different local structures of the HIV-1 immunogenic crown in various virus isolates

Local and global structural properties of the HIV-1 principal neutralizing epitope were studied in terms of NMR spectroscopy data reported in literature for the HIV-Haiti and HIV-RF isolates. To this effect, the NMR-based method comprising a probabilistic model of protein conformation in conjunction with the molecular mechanics and quantum chemical computations was used for determining the ensembles of conformers matching the NMR requirements and energy criteria. As a matter of record, the high resolution 3D structure models were constructed for the HIV-Haiti and HIV-RF immunogenic crowns, and their geometric parameters were collated with the ones of conformers derived previously for describing the conformational features of immunogenic tip of gp120 from Thailand and MN HIV-1 strains. The HIV-1 neutralization site was demonstrated to constitute in water solution highly flexible system sensitive to its environment. This inference is completely valid for the geometric space of dihedral angles where statistically significant differences in local structures of simulated conformers have been found for all virus isolates of interest. In spite of this fact, the stretch analyzed was shown to manifest a certain conservatism in the space of atomic coordinates, building up in four HIV-1 isolates two spatial folds similar to those observed in crystal for the V3 loop peptides bound to different neutralizing Fabs. The results are discussed in the light of literature data on HIV-1 neutralizing epitope structure.     

Structure and Polymorphism of the Principal Neutralization Site of Thailand HIV-1 Isolate

Abstract

Refining the geometric parameters for the ensemble of conformers, derived earlier in terms of NMR-spectroscopy data for the immunogenic tip of Thailand HIV-1 isolate, was carried out by quantum chemical methods. As a result, (i) the energy characteristics of initial structures were significanly improved, (ii) their relative locations on the scale of formation heats were determined, and (iii) the energy barriers between conformers under study were computed. On the basis of all data obtained, the high resotion 3D structure model, describing the set of stable conformers and containing the biologically active conformation, was proposed for neutralizing epitope of Thailand HIV-1 isolate. The following major conclusions were made based on the analysis of simulated conformations: i) the Gly-Pro-Gly-Gln-Val-Phe stretch forming the immunogenic crown of Thailand HIV-1 isolate exhibits the properties characteristic for metastable oligopeptide that constitutes in solution the dominant structure with other conformations admissible; (ii) three structures out of five NMR-based starting models form the cluster of conformers which adequately describes general conformational features of this functionally important site of gp120; (iii) two structures residing in this cluster are found to be well-ground for implementing the function of immunoreactive conformation of the stretch of interest; (iv) in spite of this observation, the “global” structure which gives rise to inverse γ-turn in the central Gly-Pro-Gly crest of Thailand HIV-1 gp120 is proposed to be the most probable conformation responsible for the formation of viral antigen-antibody complex in particular case under study.     

Local structural properties of the V3 loop of Thailand HIV-1 isolate

The model of locally accurate conformation for the HIV-Thailand principal neutralizing determinant (PND) located within the V3 loop of the virus envelope protein gp120 was built in terms of NMR spectroscopy data. To this end, the NMR-based conformational analysis of synthetic molecule representing the peptide copy of the fragment under study was carried out using the published sequential d connectivity data and values of spin-spin coupling constants. As a result, (i) the local structure for the V3 loop from Thailand isolate was determined, (ii) the conformations of its irregular segments were analyzed, and the secondary structure elements identified, (iii) the ensemble of conformers matching the experimental and theoretical data was derived for the stretch forming the neutralizing epitope of the HIV-Thailand PND, (iv) to estimate the probability of realizing each of these conformers in solution, the results obtained were collated with the X-ray data for corresponding segments in synthetic molecules imitating the central region of the HIV-MN PND as well as for homologous segments 39-44 in Bence-Jonce REI protein (BJRP), 41-46 in immunoglobulin lambda (Ig lambda), and 50-55 in beta-chain of horse hemoglobin (HH), (v) to find the conserved structural motifs inside diverse HIV-1 isolates, the structure determined was compared with the one derived earlier for the HIV-MN PND from NMR spectroscopy data, (vi) on the basis of all data obtained, the 3D structure model describing the set of biologically relevant conformations, which may present different antigenic determinants to the immune system in various HIV-1 isolates, was proposed for the immunogenic crown of the V3 loop. The results obtained are discussed in conjunction with the data on the structure for the HIV-1 PND reported in literature.     

Determining the invariant structure elements of the HIV-1 variable V3 loops: insight into the HIV-MN and HIV-Haiti isolates

The computer approaches that combined the 3D protein structure modeling with the mathematical statistics methods were used to compute the NMR-based 3D structures of the HIV-1 gp120 V3 loop for the HIV-MN and HIV-Haiti isolates in water as well as to compare their conformational characteristics with the purpose of determining the structure elements common for the two virus modifications. As a result, the variability of the amino acid sequence was found to stimulate the considerable structural rearrangements of the V3 loop. However, despite this fact, one functionally crucial stretch of V3 and a greater portion of its residues were shown to preserve the conformations in the viral strains of interest. To reveal the structural motifs and individual amino acids giving rise to the close conformations in the HIV-MN and HIV-Haiti V3 loops regardless of the sequence and environment variability, the simulated structures were collated with those deciphered previously in terms of NMR data in a water/trifluoroethanol mixed solvent. The structure elements and single residues of V3 residing in its biologically significant sites and keeping the conformations in all of the cases at question are considered to be the promising targets for anti-AIDS drugs studies. In this context, the structurally inflexible motifs of V3 presenting the weak units in the virus protection system may be utilized as the most convenient landing-places for molecular docking of the V3 loop and ligand structures followed by selecting chemical compounds suitable as a basis for the design of safe and effective antiviral agents.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

RNABase: an annotated database of RNA structures

RNABase is a unified database of all three-dimensional structures containing RNA deposited in either the Protein Data Bank (PDB) or Nucleic Acid Data Base (NDB). For each structure, RNABase contains a brief summary as well as annotation of conformational parameters, identification of possible model errors, Ramachandran-style conformational maps and classification of ribonucleotides into conformers. These same analyses can also be performed on structures submitted by users. To facilitate access, structures are automatically placed into a variety of functional and structural categories, including: ribozymes, pseudoknots, etc. RNABase can be freely accessed on the web at http://www.rnabase.org. We are committed to maintaining this database indefinitely.     

Sampling of the native conformational ensemble of myoglobin via structures in different crystalline environments

Proteins sample multiple conformational substates in their native environment, but the process of crystallization selects the conformers that allow for close packing. The population of conformers can be shifted by varying the environment through a range of crystallization conditions, often resulting in different space groups and changes in the packing arrangements. Three high resolution structures of myoglobin (Mb) in different crystal space groups are presented, including one in a new space group P6(1)22 and two structures in space groups P2(1)2(1)2(1) and P6. We compare coordinates and anisotropic displacement parameters (ADPs) from these three structures plus an existing structure in space group P2(1). While the overall changes are small, there is substantial variation in several external regions with varying patterns of crystal contacts across the space group packing arrangements. The structural ensemble containing four different crystal forms displays greater conformational variance (Calpha rmsd of 0.54-0.79 A) in comparison to a collection of four Mb structures with different ligands and mutations in the same crystal form (Calpha rmsd values of 0.28-0.37 A). The high resolution of the data enables comparison of both the magnitudes and directions of ADPs, which are found to be suppressed by crystal contacts. A composite dynamic profile of Mb structural variation from the four structures was compared with an independent structural ensemble developed from NMR refinement. Despite the limitations and biases of each method, the ADPs of the crystallographic ensemble closely match the positional variance from the solution NMR ensemble with linear correlation of 0.8. This suggests that crystal packing selects conformers representative of the solution ensemble, and several different crystal forms give a more complete view of the plasticity of a protein structure.     

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.     

Explicit and implicit water simulations of a beta-hairpin peptide.

The conformational properties of a beta-hairpin peptide (YITNSDGTWT) were studied by using both explicit and implicit water simulations. The conformational space of the peptide was scanned by using a restricted hydrogen-bonding search method. The search method used generated the conformational space with enough diversity and good representation of beta-hairpin structures. By using a total surface area-based treatment of hydrophobic interactions, implicit water simulations failed to discriminate between experimental beta-hairpin structures from the rest of the conformers present in the authors' conformation library. However, with inclusion of vibrational free energy and accounting separately for polar and nonpolar surface areas, the nuclear magnetic resonance structure was ranked successfully as the most stable conformation. There is a loose correlation between the conformational energies by the continuum model and the conformational energies by explicit water simulation for conformers with similar structures. However, in terms of solvation energy, both approaches have a much better correlation. By using proper treatment of surface effect (partition of the surface area into polar and nonpolar areas) and including vibrational free-energy contribution, the continuum models should be reliable. Furthermore, the authors found that, for this peptide, beta-hairpin structures have large vibrational entropy that contributes decisively to the stability of folded beta-hairpin structures. Proteins 1999;37:73-87.     

Spatial structure of the rp142 peptide containing the immunodominant epitope of the HIV-1 gp120 protein. A theoretical study]

A model of spatial structure of the synthetic peptide rp142 (24 amino acid residues) containing the immunodominant epitope of the HIV-1 protein gp120 in the region Gly-10-Phe-15 was constructed by the method of "constrained" molecular mechanics, which uses the algorithms of theoretical conformational analysis, based on NMR spectroscopy data. A comparative analysis of calculated conformations revealed that the spatial structure of rp142 in solution can be described by a family of conformations to which nine different structural clusters involving the sets of topologically close conformers correspond. It is shown that the main chain of the peptide forms irregular but "structured" conformations in which the main portion of amino acid residues is incorporated into beta-turns and helix-like fragments, while Pro-11 and Gly-12 form in some structures inverse gamma-turns, which rarely occur in protein-peptide molecules. It was found that the spatial packing of the Gly-10-Phe-15 hexapeptide in different clusters is realized at different internal rotation angles, to which topologically close structures correspond. It is assumed that this invariant structural element describes the "conformation of complex formation" that is complementary to the antigen-binding center of antibodies and is responsible for their binding to the peptide.     

Crystal structure analysis of the exocytosis-sensitive phosphoprotein, pp63/parafusin (phosphoglucomutase), from Paramecium reveals significant conformational variability.

During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis.     

The Search for the Spatial and Electronic Requirements of a Drug

A new program called GAMMA (genetic algorithm for multiple molecule alignment) has been developed for the superimposition of several three-dimensional chemical structures. Superimposition of molecules and evaluation of structural similarity is an important task in drug design and pharmaceutical research. Similarities of compounds are determined by this program either based on their structural or their physicochemical properties by defining different matching criteria. These matching criteria are atomic properties such as atomic number or partial atomic charges. The program is based on a combination of a genetic algorithm with a numerical optimization process. A major goal of this hybrid procedure is to address the conformational flexibility of ligand molecules adequately. Thus, only one conformation per structure is necessary and the program can work even when only one conformation of a compound is stored in a database. The genetic algorithm optimizes in a nondeterministic process the size and the geometric fit of the overlay. The geometric fit of the conformations is further improved by changing torsional angles combining the genetic algorithm and the directed tweak method. The determination of the fitness of a superimposition is based on the Pareto optimization. As an application the superimposition of a set of Cytochrome P450c17 enzyme inhibitors has been performed.Electronic Supplementary Material available.     

Characterization of the bioactive form and molecular determinants of recognition of cyclic enkephalin peptides at the δ-opioid receptor

An extensive and systematic search strategy to determine the conformational profile of 12 cyclic disulfide-bridged opioid peptides with varying affinities at the δ receptor has been carried out to identify the structure that is recognized by the δ receptor for each analogue. The methods and procedures used here for the conformational search have already been validated for [D -Pen2, D -Pen5] enkephalin (DPDPE), one member of this family. Use of these methods led to a low-energy solution conformation of DPDPE in excellent agreement with all the geometric properties deduced from its solution nmr spectra. Each of the analogue was subjected to the same procedure, involving a combination of molecular dynamics simulations at high and low temperature. The study was repeated in two environmental conditions, an apolar environment, simulated by using a distance-dependent dielectric constant, and a polar environment by embedding the peptides in a high constant dielectric ( ε = 80). An automated comparison of the different conformers based on their backbone rms and average distance between the key aromatic moieties was followed by graphic analysis using maximum structural overlap. The cross-comparison of the conformations for each analogue revealed a unique conformer that may be recognized by the δ receptor for each high-affinity analogue that permitted maintaining the critical elements required for recognition in a simple spatial orientation, while maximizing similarity in other regions. © 1993 John Wiley & Sons, Inc.     

Phenylisoserine in the gas-phase and water: Ab initio studies on neutral and zwitterion conformers

The conformational landscape of phenylisoserine (PhIS) was studied. Trial structures were generated by allowing for all combinations of single-bond rotamers. Based on the B3LYP/aug-cc-pVDZ calculations 54 conformers were found to be stable in the gas phase. The six most stable conformers were further optimized at the B3LYP/aug-cc-pVTZ and MP2/aug-cc-pVDZ levels for which characteristic intramolecular hydrogen bond types were classified. To estimate the influence of water on PhIS conformation, the IEF-PCM/B3LYP/aug-cc-pVDZ calculations were carried out and showed 51 neutral and six zwitterionic conformers to be stable in water solution. According to DFT calculations, the conformer equilibrium in the gas phase is dominated by one conformer, whereas the MP2 calculations suggest three PhIS structures to be significantly populated. Comparison of DFT and MP2 energies of all 57 structures stable in water indicates that, in practice, one zwitterionic and one neutral conformer determine the equilibrium in water. Based on the AIM calculations, we found that for the neutral conformers in vacuum and in water, d(H...B) is linearly correlated with Laplacian at the H-bond critical point.     

Structural aspects of Ca2+ binding by acyclic peptides: low-energy conformational domains and molecular dynamics of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide.

We have applied random-search, energy minimization and molecular dynamics simulations to investigate the structural aspects of the interaction of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide with Ca2+. Spectral data on related peptides had suggested that the beta-turn conformation might be a prerequisite for the binding of cation ion by such short linear peptides. In order to relate the conformational characteristics with the Ca(2+)-binding affinities of these peptides, the molecular events involved in cation binding need to be understood. We have addressed this problem in this study by using a systematic approach that involved the following steps. First, a random search technique was used to generate a large population of conformers for the free peptide in the absence of Ca2+. Next, the energies of these conformers were computed. Conformations with energies within 4 kcal/mol of the global minimum were analysed and found to fall into four main groups characterized by the presence of different types of hydrogen-bonded structures including single and consecutive beta-turns. The energies for interconversion of conformers from one group to another were computed and found to be relatively small (< 10 kcal/mol). Finally, molecular dynamics of the peptide at 300K in the presence of Ca2+ were used to simulate the cation binding process. Starting points for these simulations were generated by placing the ion in the vicinity of two molecules of the peptide. The simulation results showed that the conformers with two consecutive beta-turns led to the formation of a stable 2:1 (peptide:Ca2+) sandwich complex in agreement with earlier experimental observations on similar linear peptides. While the starting conformation of the peptide in the consecutive beta-turn structure allowed for the proper orientation of three carbonyl oxygen atoms for chelation to the metal ion, the dynamics of complex formation rearranged the peptide structure substantially, leading to the formation of an 8-coordinated Ca2+ complex in a dodecahedral spatial arrangement. Thus, based on the energetics of the structures and processes involved, the present study demonstrates that: a) peptide-Ca2+ complex formation is initiated by conformers adopting consecutive beta-turn structures which subsequently go over to a significantly different conformation found in the complex; and, b) The facile interconversion between the low-energy conformers in the different groups would help shift the equilibrium population towards the consecutive beta-turn structure during the complex formation.